Abstract

Nonclinical safety assessment of biopharmaceuticals for human use relies on their cross-reactivity with the respective target in other species, the absence of which precludes testing in commonly used animal models. This is the case for a first-generation of T cell-engaging BiTE antibodies, which solely cross-react with T cells of chimpanzee. BiTE antibodies are designed to transiently connect the CD3 subunit of T cell receptors on T cells with an antigen on target cells. This can very effectively activate T cells, leading to a potent redirected lysis of target cells.

Here, we have generated a series of novel human anti-CD3 antibodies that cross-react with non-human primate species, including Cynomolgus monkeys (Macaca fascicularis) and marmoset (Callithrix jacchus). The variable domains of a selected anti-CD3 antibody were used to generate next-generation BiTE antibodies specific for (1) CD33, a target antigen for treatment of acute myelogenous leukemia, (2) melanoma-associated chondroitin sulfate proteoglycan (MCSP), a potential target for treatment of melanoma, and (3) epidermal growth factor receptor 1 (EGFR), a validated target for treatment of colorectal cancer.

In vitro, CD33-, MCSP- and EGFR-specific BiTE antibodies showed comparable binding to CD3 on human and macaque T cells, and to CHO cells expressing CD33, MCSP and EGFR of human or macaque origin. In human and macaque cell-based assays, bispecific binding triggered redirected target cell lysis as well as conditional T cell activation, as previously described for first-generation BiTE antibodies.

When continuously infused for one week to Cynomolgus monkeys, the CD33-specific BiTE antibody caused depletion of CD33-expressing peripheral blood cells at tolerated doses, and reached a maximum tolerated dose (MTD) at 120 µg/m2/day. The EGFR-specific BiTE antibody reached an MTD at 150 µg/m2/kg. In contrast, the MCSP-specific BiTE antibody was well-tolerated up to a dose of 5,000 µg/m2/day. Our primate data suggest that the tolerability and toxicity of CD33-, MCSP- and EGFR-specific BiTE antibodies were apparently determined by target cell-dependent bispecific binding, and not by monovalent binding to CD3 on macaque T cells. Our data validate the Cynomolgus monkey as a relevant species for assessing the safety and tolerability of next-generation BiTE antibodies that use a more broadly cross-reactive anti-CD3 human single-chain antibody.